WO1992000055A1 - Compositions et procedes pour la prevention et le diagnostic de la maladie de lyme - Google Patents
Compositions et procedes pour la prevention et le diagnostic de la maladie de lyme Download PDFInfo
- Publication number
- WO1992000055A1 WO1992000055A1 PCT/US1991/004056 US9104056W WO9200055A1 WO 1992000055 A1 WO1992000055 A1 WO 1992000055A1 US 9104056 W US9104056 W US 9104056W WO 9200055 A1 WO9200055 A1 WO 9200055A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- ospa
- burgdorferi
- polypeptides
- infection
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
- Y10S530/825—Bacteria
Definitions
- This invention relates to compositions and methods useful for the prevention, treatment and diagnosis of Lyme disease in humans and other animals. More particularly, this invention relates to OspA and OspB polypeptides which are able to elicit in a treated patient, the formation of an immune response which is effective to treat or protect against Lyme disease. This invention also relates to a screening method for selecting the OspA and OspB polypeptides of this invention which are able to elicit such an immune response. Also within the scope of this invention are antibodies directed against the OspA and OspB
- polypeptides and diagnostic kits comprising the
- Lyme borreliosis is the most common vectorborne infection in the United States [S.W. Barthold et al.. "A n Animal Model For Lyme Arthritis", Ann. N.Y. Acad. Sci., 539, pp. 2.4-73 (1988)]. It has been reported in every continent except Antarctica. The clinical hallmark of Lyme Disease is an early expanding skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities.
- the causative agent of Lyme disease is a recently recognized spirochete known as Borrelia burgdorferi. transmitted primarily by ixodes ticks that are part of the Ixodes ricinus complex. B. burgdorferi has also been shown to be carried in other species of ticks and in mosquitoes and deer flies, but it appears that only ticks of the I. ricinus complex are able to transmit the disease to humans.
- Lyme disease generally occurs in three stages. Stage one involves localized skin lesions (erythema migrans) from which the spirochete is
- Stage two occurs within days or weeks, and involves spread of the spirochete through the patient's blood or lymph to many different sites in the body including the brain and joints. Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent. Stage three, or late
- infection is defined as persistent infection, and can be severely disabling. Chronic arthritis, and
- ELISA enzyme-linked immunosorbent assay
- Borrelia belong to the eubacterial phylum of spirochetes [A.G. Barbour and S.F. Hayes, "Biology Of Borrelia Species", Microbiol. Rev.. 50, pp. 381-400 (1986)].
- Borrelia burgdorferi have a protoplasmic cylinder that is surrounded by a cell membrane, then by flagella, and then by an outer membrane. Embedded in the outer membrane are two major proteins, a 31 kd outer-surface protein A (OspA) [A.G.
- OspB may not be produced at all in culture [T.G. Schwan et al., "Changes In Infectivity And Plasmid Profile Of The Lyme Disease Spirochete, Borrelia Burgdorferi. As A Result Of In Vitro Cultivation", Infect. Immun.. 56, pp. 1831-36 (1988)].
- the spirochete may be able to sequester itself in certain intracellular sites where it remains unavailable to circulating antibody molecules.
- SCID mice infected with B. burgdorferi contract a chronic infection associated with arthritis and carditis, similar to Lyme disease in humans.
- U.E. Schaible et al. "The Severe Combined Immunodeficiency Mouse: A Laboratory Model For The Analysis Of Lyme Arthritis And Carditis", J. Exp. Med., 170, pp. 1427- 32 (1989)].
- This system it was shown that
- immunocompromised animals are not well suited for the study of potential vaccines. Others have attempted to infect immunocompetent strains of laboratory mice, but have failed, see S.W. Barthold et al., supra. Thus, additional animal systems and vaccine development is required.
- B. burgdorferi and related proteins which are able to elicit a protective immune response.
- an appropriate animal model which mimics the human disease is required with which to study and select such antigens, and to explore the immune response they may confer.
- the present invention solves the problems referred to above by providing in one preferred
- the preferred compositions and methods of this embodiment are characterized by OspA polypeptides which elicit in a treated patient, the formation of an immune response which is effective to treat or protect against Lyme disease as caused by infection with B. burgdorferi.
- invention provides OspB polypeptides and
- compositions and methods comprising those polypeptides, which are useful for the treatment or prevention of Lyme disease.
- the preferred compositions and methods of this embodiment are
- OspB polypeptides which elicit in a treated patient, the formation of an immune response which is effective to treat or protect against Lyme disease as caused by infection with B. burgdorferi.
- this invention provides antibodies directed against the OspA or OspB polypeptides of this invention, and pharmaceutically effective compositions and methods comprising those antibodies.
- the antibodies of this embodiment are those that are immunologically reactive with the OspA or OspB polypeptides of this invention, and are effective to treat or protect against Lyme disease as caused by infection with B. burgdorferi.
- This invention further provides a novel screening process, using a specific nonhuman, mammalian model, for selecting the preferred OspA and OspB polypeptides and antibodies of this invention that are effective to protect against Lyme disease.
- screening process of this invention comprises the steps of:
- polypeptides or antibodies which are effective to protect the animal against Lyme disease.
- OspA or OspB polypeptides provides diagnostic means and methods characterized by OspA or OspB polypeptides, or antibodies directed against these polypeptides. These means and methods are useful for the detection of Lyme disease and
- this invention provides DNA sequences that code for the OspA and OspB polypeptides of this invention, recombinant DNA molecules that are
- Figure 1 depicts the DNA and amino acid sequences of the OspA polypeptide of B. burgdorferi strain N40, and the sequence of the oligonucleotide primers used to amplify the gene.
- This invention relates to OspA and OspB polypeptides, anti-OspA and anti-OspB polypeptide antibodies, compositions containing the peptides and antibodies, and methods for the detection, treatment and prevention of Lyme disease. More specifically, in one embodiment, this invention relates to compositions and methods comprising OspA polypeptides that elicit in treated animals, including humans, an immune response which is sufficient to protect the animal for some period of time against Lyme disease-related disorders as a result of infection with B. burgdorferi.
- this invention relates to compositions and methods comprising OspB
- polypeptides that elicit in treated animals, including humans, an immune response which is sufficient to protect the animal for some period of time against Lyme disease-related disorders as a result of infection with B. burgdorferi.
- this invention relates to compositions and methods comprising anti-OspA or anti-OspB polypeptide antibodies that are effective to protect a treated animal for some period of time against Lyme disease-related disorders resulting from infection with B. burgdorferi.
- this invention relates to diagnostic means and methods for the
- OspA polypeptides which confer protection against Lyme disease are OspA polypeptides which prevent or lessen the severity, for some period of time, of any one of the disorders which result from infection with B. burgdorferi. including erythema migrans, arthritis, carditis, neurological disorders, and other Lyme disease-related disorders.
- OspA polypeptide denotes the OspA protein of SEQ ID NO: 4 and serotypic variants thereof, excluding strains ZS7 and B31; fragments containing at least 10 amino acids taken as a block from the amino acid sequence of the OspA protein of SEQ ID NO: 4 and serotypic variants thereof; and derivatives of either of the above, said derivatives being at least 80% identical in amino acid sequence to said OspA protein of SEQ ID NO: 4, serotypic variants thereof and fragments thereof.
- OspA polypeptide denotes polypeptides selected from the group consisting of: polypeptides that are
- polypeptides that are immunologically reactive with antibodies that are immunologically reactive only with the OspA proteins of strains ZS7 and/or B31);
- polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the OspA polypeptide of SEQ ID NO: 4 and serotypic variants thereof excluding ZS7 and B31, (i.e.,
- polypeptides ⁇ f strains ZS7 and/or B31
- polypeptides that elicit in a treated mammalian host an mmune response that is effective to protect against Lyme disease as caused by infection with B. burgdorferi and that are capable of eliciting antibodies that are immunologically reactive with the OspA polypeptide of SEQ ID NO: 4 and serotypic variants thereof.
- OspA OspA polypeptide
- variant is any polypeptide which may be encoded, in whole or in part, by a DNA sequence which hybridizes, at 20-27°C below Tm, to any portion of the DNA sequence encoding the OspA polypeptide of SEQ ID NO: 4.
- polypeptide is any polypeptide which may be encoded, in whole or in part, by a DNA sequence which hybridizes to any portion of a DNA sequence encoding a derivative of the OspA protein of SEQ ID NO: 4 or fragments thereof, said derivatives being at least 80% identical in amino acid sequence to the OspA protein of SEQ ID NO: 4 or fragments thereof.
- OspA polypeptides include those polypeptides encoded by DNA sequences of which any portion may be amplified by using the polymerase chain reaction and oligonucleotide primers derived from any portion of the DNA sequence encoding the OspA protein of SEQ ID NO: 4.
- serotypic variants of OspA polypeptides include those polypeptides encoded by DNA sequences of which any portion may be amplified by using the polymerase chain reaction and oligonucleotide primers derived from any portion of a DNA sequence encoding a derivative of the OspA protein of SEQ ID NO: 4 or fragments thereof, said derivatives being at least 80% identical in amino acid sequence to the sequence of the OspA protein of SEQ ID NO: .4 or, fragments .fehereof. According to one embodiment of this
- a serotypic variant of an OspA polypeptide from B. burgdorferi strain N40 is provided.
- This variant (SEQ ID NO: 10), from strain 25015, differs in amino acid sequence from the OspA polypeptide of strain N40 at 39 positions.
- the protein of SEQ ID NO: 10, fragments of at least 10 amino acids taken as a block from the amino acid sequence of SEQ ID NO: 10, or derivatives at least 80% identical in amino acid sequence to SEQ ID NO: 10 or fragments thereof are all considered to be OspA polypeptides.
- polypeptides polypeptides
- antibodies which antibodies are immunologically reactive with the protein of SEQ ID NO: 10 or fragments thereof, and polypeptides capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the protein of SEQ ID NO: 10, are also considered to be OspA polypeptides.
- probes and oligonucleotide primers derived from the DNA encoding the 25015 OspA variant SEQ ID NO: 9
- substitutions as compared to the OspA polypeptide of strain N40 may be used to isolate and clone further variants of surface proteins from other B. burgdorferi strains and perhaps from other
- OspB polypeptides which confer protection against Lyme disease are OspB polypeptides which prevent or lessen the severity, for some period of time, of any ⁇ ne oi the sorders which results from infection with B... buurr ⁇ grd eri, including erythema migrans, arthritis, carditis, neurological disorders, and other Lyme disease related disorders.
- OspB polypeptide denotes: the OspB protein of B. burgdorferi strain B31 and serotypic variants thereof; fragments containing at least 10 amino acids taken as a block from the amino acid sequence of the OspB protein of B. burgdorferi strain B31 and serotypic variants thereof; and
- OspB polypeptide denotes polypeptides selected from the group consisting of: polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with
- B. burgdorferi which antibodies are immunologically reactive with the OspB protein of B. burgdorferi strain B31 and serotypic variants thereof; polypeptides that are capable of eliciting antibodies that are
- a "serotypic variant" of an OspB polypeptide is any polypeptide which may be encoded, in whole or in part, by a DNA sequence which hybridizes, at 20-27° below Tm, to any portion of the DNA sequence encoding the OspB,.polypeptide of SEQ ID "NO: 11.
- a "serotypic variant" of an OspB polypeptide is any polypeptide which may be encoded, in whole or in part, by a DNA sequence which hybridizes to any portion of a DNA sequence encoding a derivative of the OspB protein of SEQ ID NO: 11 or fragments thereof, said derivatives being at least 80% identical in amino acid sequence to the OspB protein of SEQ ID NO: 11 or fragments thereof.
- serotypic variants of OspA polypeptides include those polypeptides encoded by DNA sequences of which any portion may be amplified by using the
- OspB polypeptides include those polypeptides encoded by DNA sequences of which any portion may be amplified by using oligonucleotide primers derived from any portion of a DNA sequence encoding a derivative of the OspB protein of SEQ ID NO: 11 or fragments thereof, said derivatives being at least 80% identical in amino acid sequence to the sequence of the OspB protein of SEQ ID NO: 11 or fragments thereof.
- OspA and OspB polypeptides of this invention may be part of a larger protein.
- OspA and OspB polypeptides of this invention may be part of a larger protein.
- polypeptide of this invention may be fused at its N-terminus or C-terminus to another OspA polypeptide, or to a non-OspA polypeptide or combinations thereof.
- OspA polypeptides which may be useful for this purpose include polypeptides derived from SEQ ID NO: 4, SEQ ID NO: 10, and serotypic variants of either of the above.
- Non-OspA polypept useful for this purpose derived from SEQ ID NO: 11 and serotypic variants thereof, the B. burgdorferi flagella-associated protein and fragments thereof, other B. burgdorferi proteins and fragments thereof, and non-B. burgdorferi proteins and fragments thereof.
- fusion proteins comprising multiple serotypic variants of OspA and/or OspB polypeptides are constructed for use in the methods and compositions of this invention. Such proteins are effective in the prevention, treatment and diagnosis of Lyme disease as caused by a wide spectrum of B. burgdorferi isolates.
- the OspA and OspB polypeptides may also be part of larger multimeric proteins. These fusion proteins or multimeric proteins may be produced
- OspA and OspB polypeptides fused or coupled to moieties other than amino acids, including lipids and carbohydrates.
- a "protective antibody” is an antibody that confers protection against Lyme disease as caused by infection with B. burgdorferi. when used to passively immunize a naive animal.
- a "protective epitope” is (1) an epitope which is recognized by a protective antibody, and/or (2) an epitope which, when used to immunize an animal, elicits an immune response
- protective epitope may comprise a T cell epitope, a B cell epitope, or combinations thereof.
- T cell epitope is an epitope which, when presented to T cells by antigen presenting cells, results in a T cell response such as clonal expansion or expression of lymphokines or other immunostimulatory molecules.
- a T cell epitope may also be an epitope recognized by cytotoxic T cells that may affect intracellular B. burgdorferi infection.
- a strong T cell epitope is a T cell epitope which elicits a strong T cell response.
- B cell epitope is the simplest spatial conformation of an antigen which reacts with a specific antibody.
- a "therapeutically effective amount of an OspA polypeptide” is the amount required to prevent or lessen the severity, for some period of time, of any one of the disorders which result from infection with B. burgdorferi.
- a "therapeutically effective amount of an OspB polypeptide” is the amount required to prevent or lessen the severity, for some period of time, of any one of the disorders which result from infection with B. burgdorferi.
- an "anti-OspA polypeptide antibody” is an immunoglobulin molecule, or portion thereof, that is immunologically reactive with an OspA polypeptide of the present invention.
- an "anti-OspB polypeptide antibody” is an immunoglobulin molecule, or portion thereof, that is immunologically reactive with an OspB polypeptide of the present invention.
- An anti-OspA polypeptide antibody or anti-OspB polypeptide antibody may be an intact
- immunoglobulin molecule or a portion of an
- immunoglobulin molecule that contains an intact antigen binding site, including those portions known in the art as F(v), Fab, Fab' and F(ab')2. It should be
- an anti-OspA polypeptide antibody or anti-OspB polypeptide antibody may also be a protective antiibody.
- a "therapeutically effective amount of an anti-OspA polypeptide antibody” is the amount required to prevent or lessen the severity, for some period of time, of any one of the disorders which result from infection with B. burgdorferi.
- a "therapeutically effective amount of an anti-OspB polypeptide antibody” is the amount required to prevent or lessen the severity, for some period of time, of any one of the disorders which result from infection with B. burgdorferi.
- the OspA polypeptides of this invention in addition to including polypeptides corresponding to the native polypeptides (e.g., SEQ ID NO: 4 and serotypic variants thereof) include fragments and derivatives of those polypeptides and fragments.
- the fragments of such native polypeptides contain at least 10 amino acids taken as a block from the sequence of the OspA polypeptide of SEQ ID NO: 4 and serotypic variants thereof.
- the derivatives of this invention are at least 80% identical in amino acid sequence to the OspA protein of SEQ ID NO: 4 , serotypic variants thereof and fragments thereof.
- OspB polypeptides of this invention in addition to including polypeptides
- OspB and serotypic variants thereof include fragments and derivatives of those polypeptides and fragments.
- the fragments of such native polypeptides contain at least 10 amino acids taken as a block from the sequence of B31 OspB and serotypic variants thereof.
- the derivatives of this invention are at least 80%
- the preferred derivatives result when native OspA or OspB polypeptides or fragments are modified or subjected to treatments to enhance their immunogenic character in the intended recipient.
- various amino acid substitutions, modifications or deletions may be carried out during or after preparation of the
- Such derivatives of native OspA and OspB polypeptides include, for example, derivatives which may be produced by reacting free amino, carboxyl, or hydroxyl side groups of the amino acid residues present in the polypeptide. They may also include polypeptides which result from substitution of one or more amino acids with a different natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being preferred. For example the
- 3-methylhistidine may be substituted for histidine
- 4-hydroxyproline may be substituted for proline
- 5-hydroxylysine may be substituted for lysine; and the like.
- OspA and OspB polypeptides of the present invention may also be modified to increase their immunogenicity, for example by coupling to
- OspA and OspB polypeptides are small polypeptides synthesized chemically, it may be desirable to couple them to an immunogenic carrier. The coupling of course, must not interfere with the ability of either the polypeptide or the carrier to function
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- PPD purified protein derivative of tuberculin
- red blood cells tetanus toxoid
- cholera toxoid agarose beads
- activated carbon or bentonite.
- Any OspA or OspB polypeptide of the present invention may be used in the form of a pharmaceutically acceptable salt.
- Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.
- a method which comprises the steps of treating a patient in a pharmaceutically acceptable manner with a therapeutically effective amount of an OspA polypeptide, or a fusion protein or a multimeric protein comprising an OspA polypeptide of this invention, which confers protection against Lyme disease in a manner sufficient to prevent or lessen the severity, for some period of time, of any one of the disorders which result from infection with
- compositions that contain protective epitopes.
- protective epitopes may be B cell epitopes, T cell epitopes, or combinations thereof.
- a method which comprises the steps of treating a patient in a pharmaceutically acceptable manner with a therapeutically effective amount of an OspB polypeptide, or a fusion protein or a multimeric protein comprising an OspB polypeptide of this invention, which confers protection agai ⁇ rfe Lyme disease in a manner sufficient to prevent or lessen the severity, for some period of time, of any one of the disorders which result from infection with
- protective epitopes which may be B cell epitopes, T cell epitopes, or combinations thereof.
- OspA and OspB polypeptides of this invention for use in these compositions and methods are those containing both strong T cell and B cell epitopes. Without being bound by theory, we believe that this is the best way to stimulate high titer antibodies that are effective to neutralize
- Such preferred OspA and OspB polypeptides will be internalized by B cells expressing surface immunoglobulin that recognizes the B cell epitope. The B cells will then process the antigen and present it to T cells. The T cells will recognize the T cell epitope and respond by proliferating and
- lymphokines which in turn cause B cells to differentiate into antibody producing plasma cells.
- overlapping fragments of the OspA and OspB polypeptides of this invention are used.
- the polypeptides that contain B cell epitopes are identified by their ability to (1) be recognized by a protective anti-B. burgdorferi or anti-OspA or anti-OspB antibody (2) remove protective antibodies from polyclonal rabbit anti-B. burgdorferi serum or
- OspA and OspB polypeptides that contain T cell epitopes are identified in vitro by testing them for their ability to stimulate proliferation and/or cytokine production by T cell clones generated from humans of various HLA types, from the lymph nodes of C3H/He mice, or from domestic animals.
- Compositions comprising multiple T cell epitopes recognized by individuals with different Class II antigens are useful for prevention and treatment of Lyme disease in a broad spectrum of patients.
- an OspA or OspB polypeptide containing a B cell epitope is fused to one or more OspA or OspB polypeptides containing strong T cell epitopes.
- the fusion protein comprising the OspA or OspB polypeptide that carries both strong T cell and B cell epitopes is able to elicit high titer antibody responses effective to neutralize infection with B. burgdorferi.
- polypeptides containing B cell .epitopes are fused to segments of HBcAG or to other antigens which contain strong T cell epitopes, to produce a fusion protein comprising an OspA or OspB polypeptide that can elicit a high titer antibody response.
- an OspA polypeptide containing a B cell epitope may be fused to a strong T cell epitope of the B. burgdorferi OspB or flagella-associated protein.
- an OspB polypeptide containing a B cell epitope may be fused to a strong T cell epitope of the B. burgdorferi OspA or flagella-associated protein.
- an OspA or OspB polypeptide containing a T cell epitope may be fused to another B. burgdorferi protein or fragment thereof, containing a B cell epitope.
- a non- B. burgdorferi B cell epitope may be fused to a strong OspA or OspB T cell epitope of this invention, and so forth.
- fusion proteins comprising OspA and/or OspB
- polypeptides are constructed comprising B cell and/or T cell epitopes from multiple serotypic variants of
- each variant differing from another with respect to the locations or sequences of the epitopes within the OspA or OspB polypeptide.
- Such fusion proteins when used in the methods and
- compositions of this invention are particularly effective in the prevention, treatment and diagnosis of Lyme disease as caused by a wide spectrum of
- Multimeric proteins comprising an OspA or OspB polypeptide are also part of this invention.
- they consist of multiple T or B cell epitopes or combinations thereof repeated within the same molecule, either randomly, or with spacers (amino acid or otherwise) between them.
- spacers amino acid or otherwise
- polypeptides of this invention may be prepared by recombinant means, chemical means, or combinations thereof.
- OspA polypeptides may be any organic radicals.
- OspA polypeptides may be any organic radicals.
- OspA polypeptides may be any organic radicals.
- DNA encoding OspA and OspB polypeptides and serotypic variants thereof and derivatives thereof may likewise be cloned, e.g., using PCR and oligonucleotide primers derived from the DNA sequence encoding the OspA and OspB polypeptides of B. burgdorferi strains N40 or
- Such DNA may be expressed to produce other OspA and OspB polypeptides which are useful in the methods and compositions of this invention. Oligonucleotide primers as well as conserved and divergent DNA
- sequences within the OspA and OspB genes may also be used to isolate and clone other related surface
- proteins from B. burgdorferi and related spirochetes which may contain regions of DNA sequence homologous to the OspA and OspB polypeptides of this invention.
- OspA or OspB polypeptides of this invention are produced recombinantly they may be expressed in unicellular hosts. As is well known to one of skill in the art, in order to obtain high expression levels of foreign DNA sequences in a host, the sequences must be operatively linked to
- transcriptional and translational expression control sequences that are functional in the chosen expression host.
- the expression control sequences, and the gene of interest will be contained in an expression vector that further comprises a bacterial selection marker and origin of replication. If the expression host is a eukaryotic cell, the expression vector should further comprise an expression marker useful in the expression host.
- the DNA sequences encoding the OspA and OspB polypeptides of this invention may or may not encode a signal sequence. If the expression host is eukaryotic, it generally is preferred that a signal sequence be encoded so that the protein is secreted and matured from the eukaryotic host.
- An amino terminal methionine may or may not be present on the expressed OspA and OspB polypeptides of this invention. If the terminal methionine is not cleaved by the expression host, it may, if desired, be chemically removed by standard techniques.
- Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, cytomegalovirus and retroviruses.
- Useful expression vectors for bacterial hosts include known bacterial plasmids, such as
- plasmids from E.coli including col El, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages.
- Useful expression vectors for yeast cells include the 2 ⁇ plasmid and derivatives thereof.
- Useful vectors for insect cells include pVL 941.
- any of a wide variety of expression control sequences - - sequences that control the expression of the DNA sequence when operatively linked to it - - may be used in these vectors to express the DNA sequences of this invention.
- Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors.
- useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
- OspA and OspB polypeptides are inserted into the expression vector pDC 197-12 and transcribed from the lambda P_ Li promoter.
- thermolabile repressor CI857 Transcription in this system is controlled by the thermolabile repressor CI857.
- OspA or OspB polypeptides of this invention is inserted in frame into an expression vector that allows high level expression of the polypeptide as a fusion
- Such a fusion protein thus contains amino acids encoded by the vector sequences as well as amino acids of the OspA or OspB polypeptide. Expression of OspA and OspB polypeptides as fusion proteins may increase stability and/or facilitate purification.
- These hosts may include well known
- eukaryotic and prokaryotic hosts such as strains of E.coli. Pseudomonas, Bacillus. Streptomyces fungi, yeast, insect cells such as Spodopt era frugiperda
- SF9 animal cells such as CHO and mouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells in tissue culture.
- E. coli A89 or JM109 animal cells such as CHO and mouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells in tissue culture.
- Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the OspA or OspB polypeptide correctly, their fermentation or culture requirements, and the ease of purification from them of the products coded for by the DNA sequences of this invention.
- the molecules comprising the OspA and OspB polypeptides which are encoded by the DNA sequences of this invention may be isolated from the fermentation or cell culture and purified using any of a variety of conventional methods including: liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with
- OspA and OspB polypeptides of this invention may be generated by any of several chemical techniques. For example, they may be prepared using the solid-phase synthetic technique originally described by R. B. Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc.. 83, pp. 2149-54 (1963), or they may be prepared by synthesis in solution. A summary of peptide synthesis techniques may be found in E. Gross & H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques Of Peptide And Amino Acid Analysis, John Wiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis, Springer-Verlag
- these synthetic methods comprise the sequential addition of one or more amino acid residues to a growing peptide chain.
- peptide coupling agents are used to facilitate this reaction.
- peptide coupling agents suitable for the uses described herein see M. Bodansky, supra.
- a suitable, selectively removable protecting group is utilized for amino acids containing a reactive side group e.g. lysine.
- anti-OspA polypeptide antibodies as well as anti-OspB polypeptide antibodies are generated.
- Such antibodies are immunoglobulin molecules or portions thereof that are immunologically reactive with an OspA or OspB polypeptide of the present invention. It should be understood that anti-OspA and anti-OspB polypeptide antibodies include antibodies
- Anti-OspA and anti-OspB polypeptide antibodies of this invention may be generated by infection of a mammalian host with B. burgdorferi, or by immunization of a mammalian host with an OspA or OspB polypeptide of the present invention. Such antibodies may be polyclonal or monoclonal, it is preferred that they are monoclonal. Methods to produce polyclonal and monoclonal antibodies are well known to those of skill in the art. For a review of such methods, see Antibodies, A Laboratory Manual, supra. and D.E. Yelton, et al., Ann. Rev, of Biochem.. 50, pp. 657-80 (1981). Determination of immunoreactivity with the OspA or OspB polypeptides of this invention may be made by any of several methods well, known to those of skill in the art, including by immunoblot assay and ELISA.
- Anti-OspA and anti-OspB polypeptide antibodies may be used in compositions and methods for the prevention and treatment of Lyme disease as caused by infection with B. burgdorferi. Anti-OspA and anti-OspB polypeptide antibodies may also be used to identify OspA and OspB polypeptides containing
- This invention also provides an animal model in which to screen the various OspA and OspB
- polypeptides and anti-OspA and anti-OspB polypeptide antibodies of this invention for their ability to confer protection against Lyme disease.
- the screening process comprises the steps of
- C3H/He mice While any animal that is susceptible to infection with B. burgdorferi may be advantageously useful in this screening process, C3H/He mice are preferred, as they are not-enly susceptible to infection but are also susceptible to Lyme disease, as occurs in humans. Thus in C3H/He mice, the efficacy of responses to both infection and disease can be tested.
- polypeptide antibody may be accomplished by standard procedures. For a detailed discussion of such
- polypeptide if used, it will be administered with a pharmaceutically acceptable
- adjuvant such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM
- Such adjuvants may protect the polypeptide from rapid dispersal by
- the immunization schedule will involve two or more of the components of the immune system.
- the immunization schedule will involve two or more of the components of the immune system.
- administrations of the OspA or OspB polypeptide spread out over several weeks.
- OspA and OspB polypeptides or anti-OspA or anti-OspB polypeptide antibodies of this invention may then be used in a therapeutically effective amount in
- compositions and methods to treat or prevent Lyme disease which may occur naturally in humans and other animals.
- compositions of this invention may be in a variety of conventional depot forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liqui solutions or suspensions, liposomes, causules, supp itories, injectable and infusible solutions.
- solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liqui solutions or suspensions, liposomes, causules, supp itories, injectable and infusible solutions.
- the preferred form depends upon the
- Such dosage forms may include pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.
- carriers and adjuvants include, for example, RIBI, ISCOM, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer
- substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and
- Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates,
- polyoxyethylene-polyoxypropylene-block polymers polyethylene glycol, and wood wax alcohols.
- invention may also include other components or be subject to other treatments during preparation to enhance their immunogenic character or to improve their tolerance in patients.
- the OspA or OspB polypeptide may be formulated and administered to the patient using methods and compositions similar to those employed for other pharmaceutically important polypeptides (e.g., the vaccine against hepatitis).
- compositions comprising anti-OspA or anti-OspB polypeptide antibodies may be administered by a Kariety of dosage forms and regimens similar to those used for other passive immunotherapies and well known to those of skill in the art.
- it may be advantageous to couple such antibodies to toxins such as diphtheria, pseudomonas exotoxin, ricin A chain, gelonin, etc., or antibiotics such as penicillins, tetracyclines and chloramphenicol.
- Any pharmaceutically acceptable dosage route including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, may be used to administer the OspA or OspB polypeptide or anti-OspA or anti-OspB polypeptide antibody composition.
- the polypeptide or antibody may be any pharmaceutically acceptable dosage route, including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection.
- the polypeptide or antibody may be used to administer the OspA or OspB polypeptide or anti-OspA or anti-OspB polypeptide antibody composition.
- the polypeptide or antibody may be used to administer the OspA or OspB polypeptide or anti-OspA or anti-OspB polypeptide antibody composition.
- the polypeptide or antibody may be any pharmaceutically acceptable dosage route, including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection.
- the polypeptide or antibody may be any pharmaceutically acceptable dosage route, including parenteral
- any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramuscularly - - including paravertebrally and periarticularly - - subcutaneously,
- compositions of the invention are in the form of a unit dose and will usually be administered to the patient intramuscularly.
- OspA and OspB polypeptides or anti-OspA or anti-OspB polypeptide antibodies may be administered to the patient at one time or over a series of
- the most effective mode of administration and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course of the expected infection, previous therapy, the patient's health status and response to immunization, and the judgment of the treating physician. For example, the more highly immunogenic the polypeptide, the lower the dosage are necessary number of immunizations.
- the dosage: and necessary treatment time will be lowered if the polypeptide is administered with an adjuvant.
- the dosage will consist of 10 ⁇ g to 100 mg of the purified OspA or OspB polypeptide, and preferably, the dosage will consist of 100-1000 ⁇ g.
- the dosage for an anti-OspA or anti-OspB polypeptide antibody will be 0.5 mg-3.0 g.
- the OspA or OspB polypeptide is administered with an adjuvant, in order to increase its immunogenicity.
- Useful adjuvants include RIBI, and ISCOM, simple metal salts such as aluminum hydroxide, and oil based
- adjuvants such as complete and incomplete Freund's adjuvant.
- oil based adjuvant When an oil based adjuvant is used, the OspA polypeptide usually is administered in an emulsion with the adjuvant.
- E. coli expressing proteins comprising OspA and/or OspB
- polypeptides are administered orally to non-human animals to confer protection from infection and disease as caused by B. burgdorferi.
- a palatable regimen of bacteria expressing an OspA and/or OspB polypeptide of this invention may be administered with animal food to be consumed by wild mice or deer, or by domestic animals. Ingestion of such bacteria may induce an immune response comprising both humoral and cell-mediated components. See J.C. Sadoff et al., "Oral Salmonella Typhimurium Vaccine Expressing
- anti-OspA and anti-Osp ⁇ polypeptide antibodies as well, as the OspA and OspB polypeptides of this invention are useful as diagnostic agents for detecting infection with B. burgdorferi. because the polypeptides are capable of binding to antibody molecules produced in animals, including humans that are infected with
- B. burgdorferi. and the antibodies are capable of binding to B. burgdorferi or antigens thereof.
- Such diagnostic agents may be included in a kit which may also comprise instructions for use and other appropriate reagents.
- the detection means may be a fluorescent labeling agent such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as I or 51 Cr that produces gamma ray emissions, or a fluorescent labeling agent such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as I or 51 Cr that produces gamma ray emissions, or a fluorescent labeling agent such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as I or 51
- radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as 11 C, 15 O, or 13 N.
- incorporation of radioisotope-containing amino acids in the culture medium, or polypeptides may be conjugated or coupled to a detection means through activated functional groups.
- the diagnostic kits of the present invention may be used to detect the presence of a quantity of
- B. burgdorferi or anti-B. burgdorferi antiibodies in a body fluid sample such as serum, plasma or urine.
- polypeptide or anti-OspA or anti-OspB polypeptide antibody composition of the present invention is bound to a solid support typically by adsorption from an aqueous medium.
- solid matrices are well known in the art, and include crosslinked dextran; agarose; polystyrene; polyvinylchloride; cross-linked
- polypeptides or antibodies of the present invention may be used as diagnostic agents in solution form or as a substantially dry powder, e.g., in
- OspA and OspB polypeptides and anti-OspA and anti-OspB polypeptide antibodies provide much more specific reagents than those currently available for diagnosis, and thus may alleviate such pitfalls as false positive and false negative results.
- One skilled in the art will realize that it may be advantageous in the preparation of such reagents to utilize OspA and OspB polypeptides comprising epitopes from other
- B. burgdorferi proteins including the flagella-associated protein, and antibodies directed against such polypeptides.
- OspA and OspB polypeptides and anti-OspA and anti-OspB polypeptide antibodies of the present invention may also be useful for detection, prevention, and treatment of other infections caused by spirochetes which may contain surface proteins sharing amino acid sequence or conformational similarities with the OspA or OspB polypeptides of the present invention.
- spirochetes include Borrelia Hermsii and BorreliaRecurientis, Leptospira, and Treponema.
- mice B. burgdorferi infection and/or Lyme disease, by testing numerous strains of inbred mice. We chose to use mice because of the extensive immunologic, biologic and genetic parameters available for manipulation.
- mice We examined the susceptibility of various strains of mice to infection with the highly virulent N40 strain of B. burgdorferi. inoculated via several different routes. [S.W. Barthold et al., J. Inf.Dis., 162, pp. 133-138 (1990).] We chose mice having maximum genetic disparity and representing different H-2 haplotypes. The mice used for these studies were
- mice Balb/cByJ, C3H/HeJ, C57BL/6J, SJL/J, and SWR/J mice, purchased from the Jackson Laboratory (Bar Harbor,
- mice were housed in Micro-Isolator cages (Lab Products, Maywood, NJ) and provided food (Agway, Syracuse, NY) and water ad libidum.
- ⁇ BSK-II ⁇ BSK-II medium
- A.G. Barbour et al., Yale Journal Of Biol. Med., 57, pp. 521-25 (1986) at 34°C, to a concentration of approximately 1 ⁇ 10 3 viable (spiralling) organisms/ml, as determined by counting and on a hemacytometer using dark field microscopy.
- mice We inoculated the various strains of mice with doses of spirochetes ranging from 1 ⁇ 10 1 to 1 ⁇ 10 8 at 3 days or 3 weeks of age, via
- intraperitoneal intradermal, intragastric, and
- mice were sacrificed with carbon dioxide gas and exsanguinated by cardiocentesis.
- Tissues were homogenized in 1 ml of BSK II medium, then a 0.5 ml aliquot of the homogenate was placed in 7 ml of BSK II medium and cultured for 2 weeks.
- metatarsus, and phalanges from the infected mice for histopathology by immersion fixing these tissues in neutral-buffered formalin (pH 7.2).
- neutral-buffered formalin pH 7.2
- the ELISA was performed according to standard procedures using microtiter plates from Dynatech, Inc. coated with 0.1 ml of antigen at a concentration of 10 ⁇ g/ml, as described supra.
- the second antibody was labeled peroxidase goat anti-mouse IgG (Tago,
- mice upon intradermal infection with all doses ranging from 1 ⁇ 10 1 - 1 ⁇ 10 8 B. burgdorferi strain N40, develop clinical symptoms of a disease that is remarkably similar to Lyme disease in humans.
- Mice inoculated intradermally at 3 weeks of age developed spirochetemia and severe arthritis within two weeks. A high proportion of infected mice also developed
- C3H/He mice had high levels of anti-B. burgdorferi antibodies, and spirochetes were culturable from the spleens from day 3 to 5 after inoculation.
- C3H/He mice remained persistently infected for at least 12 months after inoculation, and had a 100% correlation between
- mice sacrificed the mice after two weeks and analyzed their blood, spleens and joints as described supra.
- mice had developed spirochetemia and severe arthritis at their ankle joints, while in all of the passively immunized mice, arthritis was prevented and spirochetes could not be cultured from their blood or spleen.
- C3H/He mice could be passively immunized against Lyme disease by transfer of polyclonal anti-B. burgdorferi serum from infected C3H/He mice.
- mice were administered 0.1 ml of the serum intradermally to groups of five uninfected C3H/He mice. Control groups of mice were immunized with normal mouse or rabbit serum. One day after immunization, we inoculated the mice intradermally on the contralateral side with 1 ⁇ 10 4 B. burgdorferi strain N40 or strain B31. After 5 or 14 days, the mice were euthanized by exposure to carbon dioxide. We then removed
- mice had positive spirochete cultures, while at least 1 to 100 spirochetes were detected in all of the control mice immunized with normal mouse or rabbit serum. Furthermore, the protective effect of the rabbit serum was maintained at a dilution of 1:500.
- Genomic DNA was purified by SDS lysis and phenol-chloroform extraction as described in F. Hyde and R. Johnson, "Genetic Relationship Of The Lyme Disease Spirochetes To Borrelia, Treponema. And Leptospira". spp. J. Clin. Micro., 20, pp. 151-54 (1984).
- oligonucleotide and protein synthesis center at Yale University.
- the sequence of the oligonucleotides was based on the known sequence of the OspA gene from
- the first member of the pair corresponded to the first 17 nucleotides of the coding sequence of the B31 OspA gene, and included an EcoR1 site and ribosome binding site at the 5' end to
- the PCR amplified OspA gene was isolated from the genomic DNA by agarose gel electrophoresis on a 1% gel, and purified by electroelution onto a DEAE
- the EcoR1-BamH1 fragment was then ligated using T4 DNA ligase (Boehringer Mannheim, Danbury, CT) into an EcoR1, BamHl cleaved expression vector pDC 197-12 (Kindly provided by W. Fiers, University of Ghent) overnight at 15°C.
- T4 DNA ligase Boehringer Mannheim, Danbury, CT
- pDC 197-12 EcoR1, BamHl cleaved expression vector pDC 197-12 (Kindly provided by W. Fiers, University of Ghent) overnight at 15°C.
- the ligation mixture was
- pDC 197-12 as the expression vector because it contains the bacteriophage lambda P L promoter and the thermolabile phage lambda cI857 repressor, which is able to completely suppress
- pDC 197-12 also contains a tetracycline resistance gene, allowing, selection of positive transformants on agarose plates containing tetracycline. We transformed the ligation mixture into competent E. coli strain A89 (Kindly provided by
- electroporation cuvette with 5 ⁇ l of the ligated DNA.
- Colonies containing the 197-OspA-N40 plasmid were identified as follows. Colonies were picked into 2 ml L-Broth with tetracycline, and incubated with shaking overnight at 30°C. The cells were then
- the sequence of the OspA gene was determined using a Sequenase kit (U.S. Biochemical, Cleveland, OH) and oligonucleotide primers synthesized at Yale University.
- the OspA gene was found to be 819 nucleotides in length.
- N40 OspA differs from B31 OspA at 2 positions, corresponding to nucleotides 117 and 446.
- the OspA protein from N40 (SEQ ID NO: 4) has an asparagine at amino acid 39 instead of a lysine, and a glutamic acid at amino acid 149 instead of glycine.
- sequence (SEQ ID NO: 3) of the N40 OspA gene was also compared to the sequence of the OspA gene from strain ZS7 [B. Wallich et al., "Cloning And Sequencing Of The Gene Encoding The Outer Surface
- Protein A Of A European Borrelia Burgdorferi
- oligonucleotide primers used to amplify the 41 kd flagella associated protein were (SEQ ID NO: 7)
- burgdorferi fusion proteins by incubating the plaques on nitrocellulose filters soaked with IPTG, following the manufacturer's protocol.
- the fusion proteins bind to the filters, which are then screened by incubation with polyclonal anti-B burgdorferi antiserum from C3H/He mice infected with B. burgdorferi. as described supra.
- We detect binding of the primary antibody by subsequent binding of alkaline phosphatase conjugated goat anti-mouse secondary antibody.
- the alkaline phosphatase which collects at the site of primary antibody binding will be detected by exposure to BCIP and NBT, using
- N40 OspA recombinant N40 OspA protein
- N40-OspA recombinant N40 OspA protein
- a unique band was present in lanes of extracts from induced cultures, accounting for approximately 5% of the total bacterial protein. The unique band migrated at 31 kd, the size expected for the
- burgdorferi monoclonal antibody VIIIC3.78 prepared by fusion of spleen cells from mice immunized with whole, live B. burgdorferi, infra. The strips were incubated for 1 hour at room temperature, washed for 1 hour at room temperature with PBS, incubated with a 1:5200 dilution of alkaline phosphatase labeled goat anti-mouse IgG (TAGO) and developed with nitroblue tetrazolium 5-bromo 4-chloro-indolyl phosphate. The immunoblot showed that the unique 31 kd band expressed by the E. coli stained with mouse serum as well as the monoclonal antibody, positively identifying it as N40OspA.
- TAGO alkaline phosphatase labeled goat anti-mouse IgG
- mice with N40 OspA g ⁇ ne corresponded to the sequence predicted from the DNA sequence (SEQ ID NO: 3) of the N40 OspA g ⁇ ne, providing further evidence that N4 ⁇ OspA was being expressed in the recombinant bacteria.
- SEQ ID NO: 3 the DNA sequence of the N40 OspA g ⁇ ne
- N40-OspA was able to elicit an immune response that was protective against Lyme disease.
- mice were injected with E. coli transformed with the vector, pDC197-12.
- mice were synthesizing antibody against N40-OspA.
- this blot we ran a protein extract of whole, heat killed B. burgdorferi strain N40, transferred the proteins to nitrocellulose strips and incubated the strips with a 1:100 dilution of serum from the actively immunized mice as described supra.
- a strong immune response to N40-OspA was elicited in all of the actively immunized animals. The antibody response could be detected by immunoblot to a dilution of 1:1000.
- mice were infected intradermally with 1 ⁇ 10 4 B. burgdorferi strain N40, B31, or CD16, prepared as described supra. The mice were then sacrificed after 5 or 14 days, and evaluated for infection and disease as described supra.
- mice that were actively immunized with E. coli expressing N40-OspA were fully protected from infection with all strains of B. burgdorferi tested, as determined from blood and spleen cultures.
- the control mice were fully protected from infection with all strains of B. burgdorferi tested, as determined from blood and spleen cultures. In contrast, the control mice
- CD16 3/5 * Expressed as number of positive cultures/total number of cultures.
- mice were fully protected from infection at 5 days suggesting that serum from mice actively immunized with OspA is sufficient to confer protection from subsequent infection with B. burgdorferi.
- T7 Jeno 10 fusion proteins Transcription of the T7-0spA fusion proteins is driven by the bacteriophage T7 promoter.
- the oligonucleotide primers used to direct the PCR are constructed so as to result in an amplified fragment which, when cloned into pGEMEX and expressed as a fusion protein, maintains the correct reading frame of the OspA protein.
- E.coli JM 109 We transform E.coli JM 109 with recombinant pGEMEX plasmids by electroporation, as described supra.
- E. coli JM109 as the host, because it contains the gene for T7 RNA polymerase under the IPTG induced lac uv5 promoter.
- T7 RNA polymerase under the IPTG induced lac uv5 promoter.
- the T7-0spA fusion protein produced in this manner is insoluble, and can be easily purified by recovery of the insoluble pellet fraction, followed by solubilization of the recombinant protein in
- Another way to synthesize fusion proteins is to utilize the vector pGEX-2T (Pharmacia, Piscataway, N.J.) which allows expression of inserted genes as glutathione S-transferase fusion proteins.
- OspA 1-819 recombinant fusion protein
- IPTG recombinant fusion protein
- mice We immunized mice with 20 micrograms of purified OspA 1-819 by subcutaneous injection, once per week for 3 weeks.
- mice with purified glutathione S-transferase prepared as
- Example XI We bled the mice on the fourth week and prepared an immunoblot as described in Example VII, to determine if the mice were synthesizing antibody against N40-OspA. We found that the mice immunized with OspA 1-819 produced a very strong immune response to N40-OspA, as an antibody response could be detected to a dilution of 1:64,000 by immunoblot.
- mice We then challenged the mice with 1 ⁇ 10 4
- One way to identify regions of the OspA protein that contain protective B-cell epitopes is to determine which regions of the OspA protein are
- OspA 200-819 and OspA 400-819 fusion proteins
- VIIIC3.78 is the only protective epitope in the OspA protein. Nor does it imply that the region encoding the B-cell epitope recognized by VIIIC3.78 does not also contain a T-cell epitope. However, it does illustrate one method that may be used to identify protective epitopes of the OspA protein.
- OspA fusion proteins Another way to identify regions of OspA that contain B cell epitopes is to use OspA fusion proteins to absorb antibodies from protective polyclonal serum.
- the various T7-0spA or OspA-glutathione S-transferase fusion proteins are coupled to CnBr activated Sepharose in order to construct a column, using standard
- mice After two days, the immunized mice are challenged with B. burgdorferi strain N4 and then sacrificed after two weeks as described supra. Joints are clinically and histologically examined for joint inflammation, and the spleen and blood are cultured for spirochetes. After two weeks, cultures are examined for spirochetes by dark field microscopy as described supra. We are able to determine which fusion proteins are able to elicit protective antibodies, because polyclonal rabbit serum containing antibodies which recognize such fusion proteins - - containing B cell epitopes - - will be depleted of the ability to confer protection to passively immunized mice.
- mice We immunized mice with 10 ⁇ g of either OspA 400-819 or OspA 1-819 in complete Freund's adjuvant, and boosted with another 10 ⁇ g in incomplete Freund's adjuvant twice at 10 day intervals. Fourteen days later, we challenged the mice with 1 ⁇ 10 4
- mice with positive blood and/or splenic culture *** Expressed as number of mice with positive blood and/or splenic culture/
- mice Example XV - Active Immunization of C3H/He mice
- OspA fusion proteins which are found to react with protective monoclonal antibodies or which deplete the polyclonal rabbit serum of protective antibodies, are used to actively immunize C3H/He mice.
- mice with E. coli expressing the recombinant OspA fusion protein were immunize mice with E. coli expressing the recombinant OspA fusion protein, as described in
- Example VIII The antibody responses elicited by both methods of immunization are compared for optimal production. We then infect the immunized mice at 14, 30 and 94 days following immunization, and sacrifice them 5, 14, or 60 days later. We evaluate the mouse joints for inflammation and culture the spleens and blood for spirochetes. We then compare the protective effect of the purified antigen and the E. coli expressing antigen.
- Antibodies elicited in the actively immunized mice that are directed against epitopes that are shared among various strains of B. burgdorferi. will confer protection against infection with these various strains of
- B. burgdorferi To determine which epitopes of N40-OspA are able to elicit such antibodies, we immunize C3H/He mice with the various OspA fusion proteins, and challenge the mice with various strains of B. burgdorferi as described supra. We have isolated over 200 B. burgdorferi specimens from New England. We inject these various specimens into C3H/He mice to determine which are infective and/or arthritogenic, and then inoculate the various infective strains into mice which have been actively immunized with OspA fusion proteins. We design a vaccine around the epitopes that are shown to confer protection against infection with many different strains of B. burgdorferi.
- Tables VII and VIII show the results of experiments conducted to determine whether immunization with recombinant OspA would protect mice from infection with other strains of B. burgdorferi.
- N40-OspA * Expressed as number of mice with positive blood and/or
- mice were immunized with 10 ⁇ g OspA 1-819 and boosted twice, as described supra. We then
- T cell lines are generated. These T cell lines are then cloned using limiting dilution and soft agar
- the T cell clones are stimulated with the OspA fusion proteins and syngeneic antigen presenting cells.
- T cell epitopes of OspA polypeptides recognized by human T cells we isolate T cell clones from B. burgdorferi-infected patients of multiple HLA types. T cell epitopes are identified by stimulating the clones with various OspA fusion
- T cell epitopes are then correlated with Class II HLA antigens such as DR, DP, and DQ.
- Class II HLA antigens such as DR, DP, and DQ.
- the correlation is performed by utilization of B
- lymphoblastoid cell lines expressing various HLA genes.
- T cell clone When a given T cell clone is mixed with the appropriate B lymphoblastoid cell line and an OspA polypeptide, the B cell will be able to present the OspA polypeptide to the T cell. Proliferation is then measured by 3 H- Thymidine incorporation.
- recombinant proteins comprising these epitopes as well as the B cell epitopes recognized by neutralizing antibodies, for example those in Example X.
- These fusion proteins by virtue of containing both T cell and B cell epitopes, permit antigen presentation to T cells by B cells expressing surface immunoglobulin.
- T cells stimulate B cells that express surface immunoglobulin, leading to the production of high titer neutralizing antibodies.
- OspA fusion proteins by linking regions of N40-OspA known to contain B cell epitopes to strong T cell epitopes of other antigens.
- the oligonucleotide is then ligated to the 5'and 3' ends of segments of DNA encoding the B cell epitopes recognised by neutralizing antibodies, as in Example X.
- the recombinant DNA molecules are then used to express a fusion protein comprising a B cell epitope from OspA and a T cell epitope from the core antigen, thus allowing production of a strong humoral immune response against B. burgdorferi.
- flagellin protein of Salmonella Bacterial flagellin are potent stimulators of cellular and humoral
- mice immunized with this agent are challenged with B. burgdorferi as described supra.
- OspA fusion proteins comprising B cell epitopes from OspA and T cell
- OspA fusion proteins comprising T cell epitopes from OspA and B cell epitopes from OspB and/or the flagella-associated protein or other B. burgdorferi proteins. Construction of these fusion proteins is accomplished by recombinant DNA techniques well known to those of skill in the art. Fusion proteins and antibodies directed against them, are used in methods and compositions to detect, treat, and prevent Lyme disease as caused by infection with B. burgdorferi.
- B. burgdorferi strain 25015 was kindly provided by John Anderson.
- SEQ ID NO: 9 encoding a serotypic variant of an OspA polypeptide, using oligonucleotide primers (SEQ ID NO: 1 and SEQ ID NO: 2) and PCR amplification, as described in
- Example III We then sequenced this gene using the Sequenase Kit, as described in Example IV.
- SEQ ID NO: 9 the gene encoding this serotypic variant from strain 25015 was found to be 819 nucleotides in length.
- SEQ ID NO: 9 encodes a protein of 273 amino acids (SEQ ID NO: 10) .
- the 25015 OspA variant migrates by SDS-PAGE at 32.5 kd rather than 31 kd.
- SEQ ID NO: 4 the sequence of the N40 OspA protein
- the OspA variant expressed by strain 25015 may represent a novel class of surface proteins, and/or the surface protein from a novel class of
- mice with B. burgdorferi strain 25015 mice with B. burgdorferi strain 25015
- B. burgdorferi strain 25015 To produce polyclonal rabbit anti-B. burgdorferi N40 antiserum, we inoculated New Zealand white rabbits with 30 ⁇ g of extract from killed B. burgdorferi (approximately 1 ⁇ 10 7
- mice were immunized with normal rabbit serum. After 17 hours we challenged the mice with 1 ⁇ 10 4
- mice B. burgdorferi strain 25015. Two weeks later, we evaluated the mice for spirochetemia and disease. All of the immunized mice were positive for infection and disease, indicating that passive immunization with rabbit anti-B. burgdorferi strain 25015 serum does not confer protection from infection with strain 25015.
- mice We inserted SEQ ID NO: 9, encoding the 25015 OspA variant, into the vector pGEX-2T using EcoR1 and BamH1 linkers as described in Example XI. We then expressed the variant as a glutathione S-transferase fusion protein, and purified the recombinant fusion protein on a glutathione sepharose 4B column, as described previously.
- Example XXII Active immunization of mice with
- mice were immunized with glutathione
- mice with OspA For example, we immunized 20 mice with OspA from
- mice were boosted and then challenged 14 days later with 1 ⁇ 10 4 B. burgdorferi strain 25015, as described previously. Control mice were immunized with either glutathione S-transferase or E. coli transformed with the pDC 197-12 vector without insert. As shown in Table X, mice immunized with OspA from B. burgdorferi strain N40 were not effectively protected from infection with B. burgdorferi strain 25015.
- mice we immunized five mice with 10 ⁇ g of the 25015 variant fusion protein as described supra.
- N40-OspA contains epitopes not shared by the 25015 variant, and similarly, the 25015 variant contains epitopes not shared by N40 OspA.
- Example XXIII Construction of an OspA polypeptide from N40-OspA and the 25015 variant
- protective epitopes within 25015OspA by producing overlapping fragments of the protein and testing each the for presence of T cell and B cell epitopes, and/or for the ability to confer protection against Lyme disease in our animal model system.
- Such fusion proteins confer protection against a broad range of B. burgdorferi isolates.
- N40 OspB B. burgdorferi strain N40 using the Sequenase Kit, as described in Example IV.
- the gene was found to be 888 nucleotides in length.
- the OspB mRNA from strain N40 has a stop codon (UAA) at the position corresponding to amino acid 176 instead of the Glu (GAA) found in B31-OspB.
- UAA stop codon
- GAA Glu
- mice were immunized mice with 10 ⁇ g of purified B31- OspB glutathione S-transferase fusion protein in complete Freund's adjuvant and boosted 3 times at 10 day intervals with 10 ⁇ g OspB fusion protein in
- mice 14 days after immunization challenged the mice 14 days after immunization with various doses of B. burgdorferi strain N40 or B31, and evaluated for infection and disease at 14 days.
- mice readily developed spirochetemia. In contrast, the majority of mice immunized with OspB were protected from infection. Animals inoculated with 1 ⁇ 10 2 or 1 ⁇ 10 3 spirochetes were also protected from disease, with the exception of one mouse that developed mild
- polypeptides including fusion proteins and multimeric proteins that are able to confer full protection from Lyme disease as caused by infection with
- mice B. burgdorferi.
- mice We used 0.1 ml of this suspension to orally inoculate C3H/He mice. Inoculation was performed by gavage using a ball tipped metal needle. We boosted the mice with the same amount of bacteria on days 10, 20, 30 and 40. Control mice were inoculated in a similar fashion with bacteria lacking the pl97-0spAN40 plasmid. We bled the mice 7 days after the second and fourth boosts and conducted immunoblots on extracts of B. burgdorferi. as described in Example VII. The sera obtained after the second boost was diluted 1:100. Sera obtained after the fourth (last) boost was diluted 1:100, 1:500, 1:1,000, 1:5,000 and 1:10,000.
- Antibodies were detectable by immunoblot in sera obtained at both time points.
- the antibody titer obtained after the second boost was somewhat weaker than that obtained in animals immunized with a similar schedule of i.p. injections of 1 ⁇ 10 6 E. coli
- the sera obtained after the fourth boost contained antibodies detectable at a dilution of 1:5000, indicating the mice had mounted a strong humoral immune response to N40-OspA by that time.
- mice Fourteen days after the last boost, we challenged the mice by intradermal inoculation with 1 ⁇ 10 4 B. burgdorferi strain N40 and evaluated for
- mice orally vaccinated with E. coli expressing N40-OspA were protected from infection and disease. In addition, the mice showed no evidence of bacteremia.
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- 1991-06-13 SG SG1996001704A patent/SG47447A1/en unknown
- 1991-06-13 CA CA002084413A patent/CA2084413A1/fr not_active Abandoned
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1994
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1995
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Also Published As
Publication number | Publication date |
---|---|
JPH06501382A (ja) | 1994-02-17 |
US6344552B1 (en) | 2002-02-05 |
SG122738A1 (en) | 2006-06-29 |
EP0536335A1 (fr) | 1993-04-14 |
US6197301B1 (en) | 2001-03-06 |
EP0536335A4 (en) | 1993-07-14 |
CA2084413A1 (fr) | 1991-12-16 |
US5747294A (en) | 1998-05-05 |
SG47447A1 (en) | 1998-04-17 |
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